The vocal fold (VF) mucosa is a complex layered structure comprised of a squamous cell epithelium, basement membrane and lamina propria rich in extracellular matrix (ECM). Each mucosal layer holds a distinct set of functions that are together responsible for VF immune, transport and barrier capabilities, an ability to absorb considerable impact stress, and favorable viscoelastic properties allowing self-sustained tissue oscillation for voice production. Disruption or destruction of the VF mucosa due to trauma or disease has severe consequences for VF oscillation and generally results in a refractory dysphonia. Previous work in the area of VF tissue repair and regeneration has focused on homogenous biomaterials, individual cell populations, and individual or small groups of genes/proteins; however, these approaches are inadequate in attempting to reconstruct the inherent complexity of this dynamic biological and biomechanical system. A clinically meaningful approach to the replacement of large VF mucosal deficits requires a tissue engineering strategy that incorporates a layered structure, sophisticated ECM network and multiple cell types. The proposed research directly confronts these challenges by focusing on the optimization of a multi-layered VF mucosa substitute based on decellularized VF ECM and coculture of VF fibroblasts and keratinocytes. We will conduct this research using a novel combination of tissue engineering, cellular and molecular biology, proteomic/glycomic, and functional vibratory techniques; allowing us to directly evaluate cell behavior, the system-wide status of the entire ECM protein and glycan network, and the oscillatory potential of these engineered structures compared with native VF tissue. Understanding the dynamics of matrix turnover, cell- ECM interaction, and vibratory performance in a controlled in vitro environment is critical to optimizing this tissue engineering approach for future in vivo experiments and eventual clinical implementation. In addition to achieving these therapeutically driven goals, the proposed research will significantly improve understanding of the native VF mucosa at the proteomic and glycomic levels, and introduce a VF organotypic coculture model with wide-ranging applicability to the study of VF epithelial morphogenesis and development, physiologic, immunologic and barrier function.